Functional characterization of a humanized anti-Clever-1 antibody targeting monocytes and anti-inflammatory macrophages

Abstract

Targeting the immunosuppressive tumor microenvironment has gained interest in opening new avenues to fight against cancer growth. Macrophages are extremely potential candidates for targeted therapies due to their highly plastic phenotype and abundance in various tumors. Clever-1 is a multifunctional molecule expressed by a subset of human monocytes and anti-inflammatory macrophages and its expression is increased in immunosuppressive events like cancer. Genetic and immunotherapeutic depletion of Clever-1 has been shown to reduce tumor growth and metastasis in mice. However, the mechanism how Clever-1 controls cancer is largely unclear. In this study, the mechanism of action of a humanized anti-Clever-1 antibody, FP-1305, on human monocytes and macrophages was investigated to understand how Clever-1 targeting regulates macrophage and monocyte phenotype. The antibody mediated responses were measured as changes in surface marker expression by flow cytometry, cytokine secretion by ELISA and signaling pathway activation by Western blotting after treatment with FP-1305 and compared to responses produced by another antibody (9-11) targeting a different epitope on Clever-1. Our results show that while FP-1305 and 9-11 did not change the surface expression of HLA-DR, PD-L1, CD115, neither phosphorylation of Nf-κB, FP-1305 induced significantly higher TNF-α secretion in macrophages after LPS stimulation compared to 9-11. Interestingly, CpG OND, a TLR9 agonist, induced rapid downregulation of Clever-1 on human monocytes compared to other TLR ligands. In conclusion, antibodies targeting distinct epitopes on Clever-1 have different potential to induce the pro-inflammatory activities of macrophages and highlight FP-1035 as potential drug candidate to reduce tumor-related immunosuppression.